Project summary. Ribosomes, responsible for decoding mRNA into proteins, are complex molecular nanomachines made up of three RNAs and over 50 proteins. The recent publication of several ribosome structures, in complex with their substrates, co-factors, and antibiotics, have greatly increased our understanding of how the ribosome functions during translation. However, very little information about ribosome biogenesis can be extracted from the inspection of the fully assembled ribosome. A major challenge hampering the study of ribosome assembly in bacteria comes from the fact that the process is highly efficient in vivo and immature intermediates do not accumulate. We have found that factors assisting ribosomal assembly represent key tools to study the process of ribosome maturation. Mutations directed at these factors slow down ribosome assembly and make it possible to isolate and characterize ribosomal intermediates. Recent studies investigating the roles of essential GTPases in ribosome assembly have shown that several GTPases are required for both 30S and 50S assembly in all three domains of life. Previous work from our laboratory has demonstrated that RbgA, YphC and YsxC are GTPases that participate in the maturation of the large ribosomal subunit in vivo in Bacillus subtilis. Although several GTPases in bacteria, archaea, and eukaryotes have been identified as participating in ribosome assembly, the precise role these proteins play in this process remains a mystery. Genetic, biochemical, and structural data support a model in which RbgA plays an essential role in coordinating the incorporation of ribosomal proteins involved in late assembly and coordinates the formation of the central protuberance and the A, P and E tRNA binding sites. Additional genetic and biochemical evidence suggests the essential GTPases YphC (EngA) and YsxC also participate in the late stages of maturation of the 50S subunit. Our working hypothesis posits that RbgA, YphC, and YsxC act in conjunction by recognizing a common late assembly intermediate to which they bind and catalyze the final steps of 50S assembly. Alternatively, they may act sequentially on distinct large subunit intermediates. To further understand how these GTPases assist the assembly of the large subunit the following specific aims are proposed: Aim 1. Characterize and compare immature intermediates from RbgA, YphC and YsxC-depleted cells. Aim 2. Establish the functional hierarchy of RbgA, YphC and YsxC binding to the assembling 50S subunit. Aim 3. Define the binding sites of RbgA, YphC and YsxC on the ribosome. The synergistic approaches of our three research groups, spanning genetics, biochemistry, quantitative mass spectrometry and cryo-electron microscopy position us for success in this project. We anticipate this work will have profound impact on the understanding of 50S subunit maturation with implications for the druggability of the bacterial ribosome biogenesis process and the development of new antimicrobials.